The modification of proteins by -N-Acetylglucosamine (O-GlcNAc) has extensive crosstalk with Ser (Thr)-protein phosphorylation to regulate signaling and gene expression in response to nutrients/stress. The hexosamine biosynthetic pathway and its endpoint product, O-GlcNAc, plays important roles in glucose toxicity and insulin resistance. O-GlcNAcylation plays a direct role in insulin signaling and insulin resistance at many points in the insulin signaling pathway, particularly on insulin receptor substrate proteins (IRS). Another key sensor of the cell's energy state is the 5'-AMP-dependent protein kinase, AMPK. AMPK isoforms are O- GlcNAcylated and many key AMPK substrates of are also regulated by O-GlcNAc. Hyperglycemia-induced and insulin stimulated O-GlcNAcylation of the transcription factor, Sp1 underlie deregulated transcription that contributes to glucose toxicity and metabolic disease. This project will elucidate molecular events leading to insulin resistance and glucose toxicity by focusing on the roles of O-GlcNAc on proteins regulating cellular metabolism in response to nutrients, including IRS-1, AMPK and Sp1. Specific Aim 1: a. Elucidate the Regulation of Insulin Receptor Substrate (IRS) by Crosstalk Between O- GlcNAcylation and Phosphorylation. b. Systematic Analysis of the Kinetics of Insulin Signaling by Concomitantly Quantifying Changes at the Individual Site Level on key proteins in the insulin signaling pathway in O-GlcNAcylation and Phosphorylation using Novel MS Methods. Specific Aim 2. Continue to Elucidate the Roles of the Crosstalk Between O-GlcNAc and the AMP- Dependent Protein Kinase (AMPK) in Cellular Regulation. a. Characterization of AMPK's modification by O-GlcNAc, including site mapping. b. Analysis of site-specific and global regulation of AMPK's activity, targeting and subcellular localization by O-GlcNAc. c. Analysis of AMPK's regulation of O-GlcNAc transferase activity, targeting and subcellular localization. Specific Aim 3. Elucidate the Roles of GlcNAcylation of the General Transcription Factor Sp1 in its Activity, Promoter Specificity, Localization, Molecular Associations and Turnover. a. Map and quantify site occupancy of O-GlcNAc and phosphate on Sp1 in different cell types and as a function of diabetic state. b. How does nutrient-mediated O-GlcNAcylation of Sp1 affect its activity in living cells? c. Do different glycoforms of Sp1 have distinct promoter specificities? d. Do different glycoforms of Sp1 have different molecular associations, or subcellular localizations or degradation rates? The results of these studies will not only elucidate novel key mechanisms underlying deregulation in diabetes and the metabolic syndrome, but also will uncover novel avenues for therapeutics.